Monocyte adherence results in the rapid transcriptional activation and mRNA stabilization of numerous mediators of inflammation and tissue repair. While the enhancer and promoter elements associated with transcriptional activation have been studied, mechanisms linking adhesion, mRNA stabilization, and translation are unknown. GRO␣ and interleukin-1␤ (IL-1␤) mRNAs are highly labile in nonadhered monocytes but stabilize rapidly after adherence. GRO␣ and IL-1␤ transcripts both contain A؉U-rich elements (AREs) in the 3 untranslated region (UTR) which have been directly associated with rapid mRNA turnover. To determine if the GRO␣ ARE region was recognized by factors associated with mRNA degradation, we carried out mobility gel shift analyses using a series of RNA probes encompassing the entire GRO␣ transcript. Stable complexes were formed only with the proximal 3 UTR which contained the ARE region. The two slower-moving complexes were rapidly depleted following monocyte adherence but not direct integrin engagement. Deadherence reactivated the two largest ARE-binding complexes and destabilized IL-1␤ transcripts. Antibody supershift studies demonstrated that both of these ARE RNA-binding complexes contained AUF1. The formation of these complexes and the accelerated mRNA turnover are phosphorylation-dependent events, as both are induced in adherent monocytes by the tyrosine kinase inhibitor genistein and the p38 MAP kinase inhibitor of IL-1␤ translation, SK&F 86002. These results demonstrate that cell adhesion and deadhesion rapidly and reversibly modify both cytokine mRNA stability and the RNA-binding complexes associated with AUF1.

Infection-induced activation of the human cytomegalovirus major immediate early enhancer/promoter has been shown to be regulated primarily by transcription factor NF-KB cis elements. However, the mechanism(s) by which human cytomegalovirus induces NF-mcB activity is unknown. A study was therefore undertaken to determine how this virus would affect normal NF-#cB regulation. Viral infection of fibroblasts resulted in the specific stimulation of promoters containing major histocompatibility complex NF-#cB cis elements fused upstream of the chloramphenicol acetyltransferase reporter gene. Electrophoretic mobility shift assays of nuclear extracts derived from mock-and virus-infected cells showed dramatic and sustained increases in DNA-binding proteins specific for these NF-KB sequences. Experiments using MAD-3 lKB, a specific inhibitor of NF-ucB, and antibodies directed against rel family members demonstrated that the induced binding activities contained p5O and p65 proteins but not c-rel. Northern analysis indicated maximal levels of p5O mRNA by 4 h postinfection, whereas p65 and MAD-3 IicB mRNA accumulation peaked at 48-72 h postinfection, suggesting different regulatory mechanisms for p5O and p65/IicB genes. Electrophoretic mobility shift assays with deoxycholatetreated cytoplasmic extracts demonstrated a 3-to 4-fold decrease in the cytosolic stores of NF-#cB binding activity by 4 h postinfection. Western blots probed with antibodies directed against MAD-3 IicB or pp4O (a protein isolated from chicken with sequence and biochemical properties similar to those of MAD-3 IncB) indicated that a cross-reactive peptide of 39 kDa was no longer detectable after 24 h postinfection. These results demonstrate that the activation and maintenance of nuclear NF-icB DNA binding and enhancer activities upon human cytomegalovirus infection occurs by multiple mechanisms.

Paclitaxel is a frontline therapy for ovarian cancer. Our laboratory has shown that paclitaxel induces IL-8, a member of the C-X-C family of chemokines, in subsets of human ovarian cancer cells. However, the critical issue concerns the biological significance of this chemokine in human ovarian cancer. To study the influence of IL-8 on tumor growth, human ovarian cancer cell lines were transfected with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. As additional evidence that IL-8 is a crucial factor in tumor growth, it was noted that ovarian cell lines in which constitutive IL-8 expression is elevated did not form tumors. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. Examination of tissue from the inoculation site revealed a dramatically elevated cellularity, containing neutrophils and macrophages, in mice receiving IL-8-expressing tumor cells. These results suggest that IL-8 production by human ovarian tumor cells can play a role in reducing the rate of tumor growth; this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. These studies indicate a role for IL-8 in ovarian cancer control and suggest that chemotherapy-induced IL-8 may have a positive role in controlling tumor growth.

Constitutive expression of mRNAs for GRO alpha, GRO beta, GRO gamma, and MCP-1, belonging to the chemokine family of 8- to 10-kDa cytokines with chemotactic properties for granulocytes and monocytes, has been identified in freshly isolated human nasal and bronchial epithelium, and in bronchoalveolar macrophages (AM). Expression of GRO alpha, GRO gamma, and MCP-1, but not GRO beta, was found in airway epithelial cells. AM expressed all three GRO genes in addition to MCP-1. On reverse transcription, chemokine mRNAs yielded 0.5-30 cDNA molecules/cell, depending on the chemokine and cell type, as determined by a semiquantitative reverse transcriptase-polymerase chain reaction technique. When chemokine mRNA expression in AM and bronchial epithelium from healthy nonatopic individuals was compared, AM expressed more GRO alpha, but similar levels of GRO gamma, MCP-1, and interleukin-8 (IL-8), as in the bronchial epithelial cells. Modulation of chemokine expression by tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) or endotoxin [lipopolysaccharide (LPS), 100 ng/ml] exposure was studied in primary nasal epithelial cell and alveolar macrophage cultures. In epithelial cells, LPS did not induce chemokine expression but GRO alpha, IL-8, and MCP-1 were upregulated approximately 100-fold by TNF alpha; GRO gamma expression was elevated only 1.5- to 4-fold. In AM cultures, all three GROs were strongly induced by LPS with peak mRNA expression 24 h after stimulation (approximately 50- to 100-fold increase compared with control cultures). MCP-1 mRNA expression, on the other hand, was not increased by LPS in AM. GRO protein was present in supernatants of stimulated epithelial cells and AM.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of the impaired balance between pro-and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-lra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-lra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-lra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-lra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-lra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.